Currently, surgeons mainly rely on palpation and visual inspection due to the difficulties of translating findings of nuclear medicine to the operating room due to the general lack of landmarks to orient the prior nuclear medicine results. Therefore, there is a critical need for new intraoperative imaging modalities that can provide accurate, real-time, intraoperative assessment of both molecular and anatomical information resulting in identification of viable tumor, fibrotic (scar) tissue, non-malignant tissue, and tumor margins at greater depths than are available with infrared-fluorescent guided surgery as well as better resolution than endoscopy. To overcome these limitations, our objective is to develop, characterize, and validate ultra-acidic pH-responsive optoacoustic dye (pHO dye) detectable by multispectral optoacoustic tomography (MSOT) and provide real-time color maps of tissue pH to allow for identification of regions of tumor and non-malignant tissue in mice. Three innovative aspects in this proposal are: 1) development, characterization, and evaluation of the first zwitterionic optoacoustic contrast agent, prototype pHO dye, that modulates spectra in response to ? 0.2 pHe of the tissue microenvironment; 2) use of unique instrumentation, multispectral optoacoustic tomography, which provides superior resolution, penetration depth, and the ability to visualize multiple spectra simultaneously; 3) development and validation of ratiometric algorithm for generation of real-time acidic pHe color maps based upon detection of pHO dye or V7-pHO probe via MSOT imaging. We hypothesize that optimized pHO dye and V7-pHO probe will facilitate detection of tumor and differentiate it from non-malignant and fibrous tissue with high sensitivity and specificity using multispectral optoacoustic tomography (MSOT) which utilizes a ?light-excitation and sound-emission? approach resulting in superior signal to background ratio. We will test this hypothesis by the following aims: 1) characterize, optimize, and validate pHO dye and V7-pHO probe to target acidic pHe for detection of tumor cells in vitro and in tissue phantoms; 2) assess optimized pHO dye and V7-pHO probe to facilitate detection of orthotopic tumors from non- malignant tissue in vivo using multispectral optoacoustic tomography; 3) develop ratiometric algorithms based upon the spectral modulation of the optimized pHO dye and V7-pHO probe to produce real-time pHe measurement color maps of tumors. Successful completion of this proposal will result in unprecedented improvements in detection of abdominal tumors in vivo to potentially reduce the gap between the surgeon and nuclear imaging.